HIV-1 persists indefinitely in infected individuals including those successfully being treated with highly active antiretroviral therapy (HAART). Numerous studies indicate that latently infected long-lived memory CD4+ T cells are an important source of enduring infection. A strategy that is being actively pursued to eliminate the latent arm of the infection involves developing and employing drugs that act as antagonists of viral latency. To date, the latency antagonists that have been identified tend to lack a high degree of specificity. Moreover, the mechanism(s) underlying viral latency in patients is likely multifactorial. Therefore, it may be necessary to develop combination therapies possibly in conjunction with immunotherapy to ultimately eliminate the virus from patients. Drugs, which can activate latent virus with high specificity are likely to have fewer off-target effects and might b significantly less toxic in the context of combination therapy. The ubiquitin-proteasome system (UPS) has been shown to play an important role in gene regulation. A great deal of specificity is imparted to the UPS particularly by the E3 ubiquitin ligases, of which there are more than 600 in human cells. Preliminary studies indicate that the UPS is involved in maintaining HIV-1 latency since proteasome inhibitors (PIs) can activate latent virus. This has led to the hypothesis that there is a factor positively affecting HIV-1 gene expression that is targeted for proteasomal degradation in latently infected cells. Therefore, it should be possible to identify the factor and the specific E3 ligase that directs it turnover, leading to development of drugs that can specifically inhibit ubiquitylation of the positive factor activating latent virus. The R21 phase (Aims 1 and 2) of this proposal aims at identifying the positive factor and the specific E3 ligase targeting its turnover. Upon successful completion of the R21, the R33 phase (Aims 3 and 4) would involve: 1) developing an assay suitable for high-throughput screening (HTS), 2) carrying out a HTS with a library of over 200,000 diversified low molecular weight compounds, 3) characterizing hits and selecting lead compounds, and 4) conducting lead optimization to improve the potency, selectivity, and pharmaceutical properties of lead compounds, aimed at the identification of an orally available Development Candidate.